Human immunodeficiency virus type 1 (HIV-1) infection continues to spread globally and predominantly by heterosexual contact. In order for HIV to be transmitted through sexual intercourse (heterosexual and homosexual) the virus must cross the epithelial barrier of the mucosa. Rational prophylactic strategies for controlling HIV transmission rely upon a detailed cellular and molecular understanding of the initial interactions that occur between the virus and host after sexual exposure. While advances have been made to identify the kinds of cell that are first infected, very little progress has been made to delineate and understand the earliest interaction(s) of HIV-1 virions with the mucosal surface. On uninjured mucosal membranes, the first cells encountered are CD4-negative epithelial cells. Studies of mucosal transmission (vaginal) in a non-human primate model demonstrated that the first cells infected (expressing viral RNA 18 hrs after infection) are Langerhans' cells, which are located within the mucosa, beneath the epithelial cells. Although evidence continues to accumulate that suggests other cellular molecules mediate attachment of virions to the surface of CD4-negative cells there remains a great void in our understanding of the molecular and cellular events that proceed infection of Langerhans' cells. This is due in great measure to the lack of experimental methods with sufficient sensitivity and specificity to directly analyze the interactions of HIV-1 virions with the mucosa. Our previous finding clearly demonstrated the ability to directly visualize infectious HIV-1 virions that have been labeled with GFP. We have also demonstrated that GFP+ virions can be quantitatively visualized on both cell and mucosal surfaces. Thus, we propose to conduct a detailed and dynamic analysis of HIV-1 mucosal transmission. The research proposed in this grant address questions regarding the molecular determinants (viral and cellular) of initial virus attachment, penetration of virus into or across the mucosa and virus-host cell infection. Our central hypothesis is that luminal epithelial cells play an important role in the earliest events of HIV-1 mucosal transmission. It follows that this work has significant clinical implications for providing the experimental basis to proceed rationally with new strategies for inhibiting HIV-1 transmission. To test our central hypothesis we propose: (1) To define the nature of the interaction between HIV-1 and primary epithelial cells - at the level of virus binding, entry and infection; (2) To define the host-cell and virus-associated molecular determinants which mediate the physical interaction between HIV-1 virions and primary epithelial cells; (3) To define the nature of the interaction between HIV-1 virions and epithelial cells using an organ culture system and human and monkey mucosal tissues (vaginal and intestinal) and (4) To analyze the earliest events of HIV-1 mucosal transmission in animal models.